Therapeutic Uses of Recombinant Uteroglobin
The search for improved therapeutic agents for the treatment of inflammatory, as well as fibrotic diseases, has received much attention in recent years. Neonatal Respiratory Distress Syndrome (RDS), a lung surfactant deficiency disease, is a condition of particular interest in that it is one of the major causes of mortality in premature neonates. While introduction of surfactant therapy dramatically improves survival of RDS patients, the development of chronic inflammatory and fibrotic disease in a significant percentage of this patient population is a major problem. Likewise, glomerular nephropathy and renal fibrosis leads to end stage renal failure when patients' kidneys become blocked and no longer filter the blood. Many forms of glomerular nephropathy and renal fibrosis is characterized by fibronectin deposits. In both diseases, fibronectin and collagen deposition and fibrosis render the organ nonfunctional, and eventually, unable to support life. Thus, these patients require chronic hemodialysis or kidney transplantation.
Recombinant human UG is a protein with beneficial anti-inflammatory, anti-fibrotic, anti-tumor, respiratory and immunomodulatory properties that is under development as a therapeutic agent in several clinical indications. Recombinant human UG is useful for the treatment of conditions characterized by a deficiency of UG. It is especially adapted for the treatment of pulmonary inflammatory conditions, for example neonatal respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD); for the treatment of conditions characterized by an elevation in local serum PLA2 activity, such as adult RDS (ARDS), septic shock, pancreatitis, collagen vascular diseases, rheumatoid arthritis, acute renal failure, and autoimmune uveitis; for the treatment of conditions characterized by local elevations in PLA2 activity, such as neonatal RDS/BPD, ARDS, rheumatoid arthritis, asthma, peritonitis, glomerulopathies, including hereditary Fn-deposit glomerulonephritis, and autoimmune uveitis; for the treatment of fibrotic conditions where deposition of fibronectin is a causative factor e.g., idiopathic pulmonary fibrosis, bleomycin lung, and cystic fibrosis and glomerular nephropathy, particularly familial glomeruleropathy, characterized by Fn deposits in the kidneys, which ultimately lead to renal failure, can also be treated with exogenous UG; and to methods for treating or preventing an inflammatory or fibrotic condition characterized by a deficiency of endogenous functional UG, by administering a compensating amount of rhUG.
RhUG is useful for inhibiting cellular adhesion to fibronectin, inhibits inflammatory cell and fibroblast migration on already deposited fibronectin, and inhibits the interaction between a cell and an extracellular matrix protein and/or membrane bound protein. RhUG is also especially useful for improving and/or normalizing lung function, pulmonary compliance, blood oxygenation, and/or blood pH. RhUG is particularly useful in the regulation of smooth muscle concentration in various organ systems including the respiratory system, the digestive system, the circulatory system, the reproductive system, and the urinary system. RhUG may also be used as well to regulate or reduce vascular permeability, to inhibit the migration of vascular endothelial cells and angiogenesis, and to prevent angiogenesis. Intratracheal rhUG may be used as a stem cell factor to increase lymphocyte production and/or decrease polymorphonuclear leukocyte proliferation in the long term. RhUG increases the concentration of circulating lymphocytes and/or cytotoxic T cells while decreasing the concentration of circulating polymorphonuclear leukocyte proliferation, which is especially useful for patients suffering from an autoimmune disease or allergy. Intravenous rhUG may be used as well to suppress ATP metabolism in circulating lymphocytes and to increase ATP metabolism in activated neutrophil, monocytes, macrophages, and NK cells in the short term.
Prior Art Methods for the Production of Recombinant Human Uteroglobin
There are several published methods for expressing rhUG and for purifying either native or recombinant uteroglobin, or urine protein-1, in microgram to milligram quantities for research purposes (Mantile, 1993; Miele, 1992; Singh, 1987; Jackson, 1989; Anderson, 1994; Umland, 1994; Aoki, 1996). These methods are quite varied but none are well suited to large-scale production of a protein and none address the regulatory issues required of a process for production of a pharmaceutical. Furthermore, the biological activities of these various preparations are not necessarily equivalent. For example, Nieto (1997) reported that native rabbit uteroglobin loses some of its progesterone activity upon lyophilization, while Miele and Mantile use repeated size exclusion chromatography steps and multiple lyophilizations as concentration steps during their purification process. However, end users would greatly prefer a ready-to-use product over a lyophilized product since the percentage of aggregates of rhUG increases with both lyophilization and repeated freeze thaw cycles. High levels of aggregation can adversely affect the biological activity, change the immunogenicity, or alter the potency of the final drug product. Under FDA guidelines undesirable aggregates constitute an impurity, entire lots of drug product may be rejected on the basis of high levels of aggregate within the drug product.
Problems in Development of Recombinant Therapeutics
The production of the recombinant protein-based drug substances involves the development of several processes that adhere to the guidelines set forth by the United States Food and Drug Administration (FDA) referred to as current Good Manufacturing Practices (cGMP). A process that adheres to the FDA's cGMP guidelines is compliant with cGMP. In order to sell a pharmaceutical composition or drug product in the U.S. and elsewhere, it is necessary to produce the drug product using a cGMP process.
The clinical development of a recombinant protein as a drug substance, as well as the sales and use of protein drugs, require a well-characterized and reproducible production process for the drug substance as well as a detailed characterization of that drug substance.
Recombinant proteins represent a particular challenge since their activity is dependent not only upon amino acid composition but also upon the conformation of the protein. The conformation of a protein is the overall three-dimensional structure of the protein which may be characterized on four levels. The first level is its primary structure or amino acid sequence. The second level is the protein's secondary structure and is the pattern of organization associated with short stretches of about 6–30 amino acids in the protein which form local stable structural regions such as alpha helices, beta sheets, and omega loops. The third level, tertiary structure consists of the groupings of secondary structures into units or domains within a single contiguous stretch of amino acids, representing a protein or peptide monomer. The four helical bundle or fibronectin Type III repeat are examples of tertiary structures. The fourth level, quaternary structure is present when two or more individual peptide or protein monomers combine, either covalently or non-covalently, to form a single functional unit.
Recombinant proteins present a further challenge since activity is also dependent upon surface characteristics in which charge and hydrophobic character, in addition to shape, contribute significantly to the ability of a recombinant protein to interact specifically with other biological and chemical substances in a physiological environment. Isoforms of a protein consist of small variations in conformation, surface charge and/or hydrophobic character. These variations may result from changes in temperature, or from interactions with chemicals, salts, or other biological molecules (e.g., proteins, carbohydrates, lipids, nucleic acids, etc.) in the surrounding environment, or from actual chemical modifications to individual amino acids in the protein. Different isoforms of a protein can be detected by high-resolution analytical methods such as hydrophobic interaction HPLC, mass spectrometry, capillary electrophoresis, peptide mapping, isoelectric focusing, and two-dimensional electrophoresis.
The physical form and conformation of a recombinant protein drug can be strongly influenced by the expression system in which it is produced, as well as by the process through which it is purified for use as a drug substance, for example. Likewise, the resulting isoform or isoforms of the recombinant protein product can also be strongly influenced by the expression system and process through which it is produced. The biological activity of a protein is highly dependent upon its conformation and isoform(s), not just its chemical composition. For example, a protein may be partially or completely denatured by exposure to high or low temperatures and rendered biologically inactive, yet it still retains the same sequence of amino acids. Therefore, the biological activity of a recombinant protein is dependent not only on its chemical composition, but also upon the process through which it is expressed, purified, formulated and even packaged.
Drug substances or products often have extra components in addition to the biologically active compound and its vehicle or carrier. These components are derived from the raw materials from which the drug was produced or from materials introduced as part of the purification, formulation, or final packaging processes. The drug substance is defined as the final form of purified bulk drug while the drug product is the final packaged formulation of the drug substance (e.g., the product used in the patient.) These extra components of the drug substance or product are considered impurities or contaminants and may have unintended or undesirable biological activities of their own, either alone or in combination with the drug itself. Contaminants may be defined as components that are not derived from the drug itself while impurities may be defined as components that contain some element of the drug itself (e.g., fragments, variations, isoforms, enantiomers, aggregates, etc.). Thus, the drug production process is important not only because it determines the characteristics of the drug itself, but also because it determines the level and nature of contaminants and impurities in the drug substance and drug product. It is essential, therefore, to carefully define the process in order to maintain consistent and reproducible biological activity, in vivo, of a drug substance, drug product, or pharmaceutical composition. Thus, the process through which a recombinant protein drug is produced should be sufficiently well-characterized so that it is capable of complying with pharmaceutical production regulatory guidelines in order to be commercially viable, since non-compliance results in a product that cannot be sold or used in the U.S and elsewhere.
Moreover, the biopharmaceutical production process must be sufficiently efficient and economical to be commercially viable. Purification methods that are used in the laboratory to produce small amounts of a protein for research purposes are not typically suitable for biopharmaceutical production. For example, a small scale method such as size exclusion chromatography often is not practical for larger scale production because the chromatography matrix would be crushed under its own weight in the size of column required for purification of even a few grams of protein. Furthermore, size exclusion chromatography always increases the volume of the sample, resulting in less manageable high volume purification intermediates that must be concentrated prior to the next step in the process. Therefore, it is highly desirable to avoid the use of size exclusion chromatography in a biopharmaceutical production process. Another technique frequently employed to preserve a protein pharmaceutical agent in a stable form is lyophilization. This process involves the simultaneous freeze-drying of a protein, converting it from a liquid form in which it is typically susceptible to degradation, to a dry powder form in which it can typically be stored for many months without losing biological activity. However, repeated freeze/thaw cycles increase the percentage of aggregates of rhUG, which may result in a significant change in biological activity.